RBC alloimmunization remains a common clinical problem in transfusion medicine. Alloantibodies are responsible for both immediate and delayed hemolytic transfusion reactions; leading to substantial morbidity and occasional mortality. For those patients unfortunate enough to make multiple alloantibodies, provision of compatible RBCs can be both time and resource intensive. In some cases, this can result in an inability to locate an otherwise life-saving therapy. Despite its clinical importance, the fundamental molecular signals that drive RBC immunization remain unclear. Like infectious agents, the generation of alloantibodies to RBCs requires the activation of antigen specific CD4+ T cells and B cells, where naove CD4+ T cells differentiate into T Follicular Helper cells (TFH) that are essential for complete B cell differentiation into antibody secreting cells. However, our understanding of the molecular mechanisms by which naove CD4+ T cells are induced to differentiate and express a TFH genetic program in vivo remains partial. Furthermore, the molecular factors responsible for naove T cell activation and TFH differentiation by the relatively weak stimulant of RBC transfusion are unknown. If we are to ever successfully intervene in this pathogenic process, we must first identify the cellular targets and molecular signals responsible for RBC alloantibody generation. The development of clinically relevant mouse models of RBC storage and transfusion provides a novel set of tools to address this issue. Recent work in these models demonstrated that RBC alloimmunization is enhanced by RBC storage, and that transfusion of stored RBCs significantly increases the systemic production of multiple cytokines including IL-6. We have subsequently demonstrated that i) IL-6 deficient mice are less susceptible to RBC alloimmunization in both fresh and stored blood settings, ii) IL-6 signaling activates the PI3K/AKT/mTOR signaling pathway in naove CD4+ T cells, and iii) induction of PI3K/AKT/mTOR signals correlates with increased phosphorylation of multiple members of the BAF chromatin remodeling complex in primary naove T cells. We therefore propose to identify the specific cellular targets of IL-6 in RBC alloimmunization, and further determine the molecular signals in naove CD4+ T cells induced by IL-6. The objective of our research plan is to test the specific hypothesis that the IL-6 induced by transfusion of fresh and stored RBCs drives TFH differentiation from naove CD4+ T cells. Furthermore, we hypothesize that IL-6 signaling induces phospho-modulation of the BAF chromatin remodeling complex via the action of the PI3K/AKT/mTOR signaling pathway. We will test our hypothesis by combining a well established mouse model of RBC alloimmunization and storage with recently developed mouse strains expressing a floxed allele of IL-6 Receptor (IL-6RA). In the fullness of time, we anticipate that identification of the molecular circuitry driving RBC antibody production will provide therapeutic and diagnostic targets for further clinical development.